AbstractContext: In ovaries surgically removed for fertility preservation, hormone concentrations in fluid from small antral follicles were determined. Levels were compared with those found in preovulatory follicular fluid.Objective: The objective of this study is to measure intrafollicular concentrations of anti-Müllerian hormone (AMH), inhibin-A, inhibin-B, estradiol, and progesterone.Setting: The study was set in a university hospital.Patients: Patients were 22 women suffering from a cancer disease and 16 women undergoing assisted reproduction.Interventions: Fluid from 35 follicles (diameter, 3–8 mm) was included and compared with that of 32 preovulatory follicles.Main Outcome Measures: The main outcome measures were intrafollicular concentrations of the measured hormones and their possible correlation.Results: Concentrations of AMH in small antral follicles were almost three orders of magnitude higher than in follicle fluid of preovulatory follicles, 790 ± 95 vs. 1.17 ± 0.14 ng/ml (mean ± sem), respectively. There was a significant negative correlation between estradiol and AMH in fluid from small antral follicles, whereas inhibin-A and inhibin-B were correlated positively with estradiol concentrations. Progesterone showed a similar correlation to levels of AMH but only in fluid of preovulatory follicles.Conclusions: The high expression of AMH in granulosa cells of small antral follicles actually translates into very high follicle fluid AMH concentrations. This most likely explains the correlation between serum AMH levels and the number of small antral follicles as previously demonstrated. The negative correlation between estradiol and AMH suggests that FSH down-regulates AMH expression. Thus, the microenvironment of the follicle shows profound changes with developmental stage and highlights the importance of studies to understand the mechanisms that regulate follicular growth and development during antral stages of development.